Steatohepatitis-liver cancer model animal

ABSTRACT

Fatty liver was induced by administering agents for inducing organ inflammation to experimental animals to evoke insulin resistance and by rearing them with high-fat diets. As a result, steatohepatitis was successfully induced in the animals. The animals show pathological findings similar to those of humans. By using these model animals, substances for treating or preventing diseases can be efficiently screened and the efficacy of medicinal substances can be effectively evaluated.

TECHNICAL FIELD

The present invention relates to steatohepatitis/liver cancer modelanimals, and uses thereof.

BACKGROUND ART

Previously, non-alcoholic fatty liver disease was believed to be abenign disease that does not progress. However, it was revealed thateven non-drinkers develop inflammation similar to alcoholic hepatitisand show hepatic fibrosis histology, and now the non-alcoholic fattyliver disease is known as a disease with poor prognosis. In particular,metabolic syndromes due to obesity, diabetes, or the like have beendrawing attention in recent years. It is becoming a common view thatnonalcoholic steatohepatitis (NASH) is one of such syndromes. However,the mechanism remains unclear, and effective methods and/or agents fortreating NASH have not been established. This is partly because NASH isdue to human lifestyle-related diseases, and thus appropriateexperimental animals have not been established.

For the development of effective methods and agents for treating NASH,it is essential to elucidate the pathological condition of NASH, whichprogresses to lethal diseases such as liver cirrhosis and liver cancer.However, experimental animals that are currently used as a NASH modelmouse in research include single-gene modified mice such as leptinreceptor-deficient mice (Non-patent Document 1), hepatocyte-specificPten-deficient mice (Non-patent Document 2), and retinoic acid receptora dominant-negative transgenic mice (Non-patent Document 3), and miceinduced with a special diet such as a methionine/choline-deficient diet(Non-patent Document 4). However, the human pathogenesis differs fromthat in the genetically-modified mice in which a single-gene mutation isresponsible for the development and progression of the pathologicalcondition, and is unlikely to be due to only the intake of a particularnutrient. Furthermore, insulin resistance and hepatic fibrosis cannot besimultaneously monitored in these mice. Furthermore, the ALT, an indexin serobiochemical analysis, is only slightly elevated in mice thatdevelop fibrosis, and therefore several mouse tissue slices are requiredto assess their pathological condition. Conversely, the pathologicalcondition is assumed to be different from that of human because ALT iselevated to a markedly high level. Furthermore, the pathologicalcondition recovers spontaneously after treatment. This makes itdifficult to test and assess drugs for their efficacy. Thus, fordeveloping methods and agents for treating NASH, it is desirable toestablish an experimental animal model that is compatible with the humanclinical condition.

Although various studies have been conducted, there is no experimentalanimal exhibiting pathological conditions similar to those of human.Thus, under the current circumstances, it is difficult to conductdetailed screening to elucidate the pathogenesis or to establishtherapeutic methods.

PRIOR ART DOCUMENTS

-   [Non-patent Document 1] Sahai A et al., Am J Physiol Gastroentest    Liver Physiol 287: G1035, 2004-   [Non-patent Document 2] Horie Y et al., J Clin Invest 113: 1774,    2004-   [Non-patent Document 3] Yanagitani A et al., Hepatology 40: 366,    2004-   [Non-patent Document 4] Rinella M et al., Journal of Hepatology 40:    47, 2004

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An objective of the present invention is to provide steatohepatitismodel animals and liver cancer model animals that exhibit pathologicalfindings similar to those of humans, and uses thereof. Morespecifically, an objective of the present invention is to providetechniques for producing model animals that develop into fatty liver,steatohepatitis, hepatic fibrosis, liver cirrhosis, and liver cancerfrom insulin resistance.

Means for Solving the Problems

The present inventors conducted dedicated studies to achieve theabove-described objectives. The present inventors induced insulinresistance by administering an agent for inducing organ inflammation,and induced fatty liver in mice by feeding them with a high fat diet. Asa result, the present inventors successfully developed steatohepatitisin mice. A detailed observation of the mice revealed the followingpathological findings:

-   (1) macrovesicular fat deposition in liver cells and liver cell    ballooning;-   (2) infiltration of inflammatory cells; and-   (3) fibrosis around mainly the central vein.

The above-described pathological findings are characteristic of humannonalcoholic steatohepatitis (NASH). Specifically, by using theabove-described method, the present inventors for the first timesuccessfully produced mice that exhibit similar pathological findings asthose of human NASH.

The NASH animal model produced by the present inventors is differentfrom conventional animal models in the following points:

-   (1) similarly to that observed in the histophathological findings of    human, fatty degeneration and fibrosis of liver cells progress    mainly around the central vein instead of the portal vein; and-   (2) the histophathological images of human “burned-out NASH” are    observed, in which only hepatic fibrosis is seen, although fat    deposition and loss of inflammatory cells are observed as the    pathological condition progresses.

The animal model of the present invention is characteristic in that itis produced without genetic modification. Furthermore, the model animalsof the present invention unfailingly (100%) develop a pathologicalcondition similar to the progression and prognosis of human NASH at aconstant time course, and are the first model animals that exhibit thesame course of progression of pathological condition in human. Inaddition, the model animals of the present invention have a remarkablybeneficial effect in that all of insulin resistance, fatty liver,steatohepatitis, hepatic fibrosis, and liver cirrhosis can be observedat the same time.

Moreover, the present inventors newly found that the above-describedNASH model animals develop liver cancer following liver cirrhosis asthey continue the rearing. This is the same change of pathologicalcondition observed in humans. Thus, the animals prepared by the methodsof the present invention are very useful as model animals of human livercancer.

Human liver cancer causes the liver surface to bulge. However,conventional model animals for liver cancer, which are prepared byadministering a chemical substance, develop liver cancer that does notcause bulging of the liver surface. Meanwhile, model animals of thepresent invention, which are prepared without genetic modification anddrug administration, exhibit bulging of the liver surface in a fashionsimilar to the case of human liver cancer. Thus, the model of thepresent invention is a model much closer to human liver cancer.Furthermore, liver cirrhosis is not developed by administering chemicalsubstances, while model animals of the present invention develop massivetype cord-like liver cell carcinoma. Furthermore, infiltration ofinflammatory cells and development of liver cancer caused by cirrhosisto displace normal liver cells are observed in the model animals of thepresent invention. In the model, the origin of liver cancer ismacrovesicular fatty liver which shows a pathological condition verysimilar to that of human NASH. The liver cancer develops from hepaticfibrosis and liver cirrhosis. Thus, the animal model of the presentinvention is very useful, as it has not been reported previously.

As described above, the present inventors successfully produced modelanimals for steatohepatitis and liver cancer which show similarpathological findings to those of human, and thereby completed thepresent invention. By using these model animals, it is possible toefficiently screen for substances for treating or preventing diseases,and effectively evaluate the efficacy of medicinal substances.

The present invention relates to model animals which develop into fattyliver, steatohepatitis, hepatic fibrosis, cirrhosis, and liver cancerfrom insulin resistance, and more specifically, the present inventionprovides:

-   [1] A non-human animal model for steatohepatitis produced by    administering an agent for inducing organ inflammation;-   [2] The non-human animal of [1], wherein the steatohepatitis is a    non-alcoholic steatohepatitis;-   [3] a non-human animal model for diabetes produced by administering    an agent for inducing organ inflammation;-   [4] the non-human animal of any one of [1] to [3], wherein the agent    for inducing organ inflammation is an N-acetyl-β-D-glucosaminidase    inhibitor;-   [5] the non-human animal of any one of [1] to [4], which comprises    the step of inducing fatty liver by administering an agent for    inducing organ inflammation to the animal and rearing the animal    with a high-fat diet;-   [6] the non-human animal of any one of [1] to [5], wherein the    non-human animal is a mouse;-   [7] a method of producing a non-human animal model of    steatohepatitis, which comprises the step of inducing inflammation    in an organ of the non-human animal;-   [8] a method of screening for a substance for treating or preventing    steatohepatitis, which comprises the steps of:-   (a) administering a test substance to the non-human animal model of    steatohepatitis of [1]; and-   (b) evaluating an ameliorating effect on steatohepatitis;-   [9] a method of evaluating a medicinal substance for efficacy    against steatohepatitis amelioration, which comprises the steps of:-   (a) administering a test medicinal substance to the non-human animal    model of steatohepatitis of [1]; and-   (b) evaluating an ameliorating effect on steatohepatitis;-   [10] a method of screening for a substance for treating or    preventing a diabetic disorder, which comprises the steps of:-   (a) administering a test substance to the non-human animal model for    a diabetic disorder of [3]; and-   (b) evaluating an ameliorative effect on diabetic disorder;-   [11] a method of evaluating the side effects risks of a    pharmaceutical agent for treating or preventing a diabetic disorder,    which comprises the steps of:-   (a) administering a test pharmaceutical agent to the non-human    animal model for a diabetic disorder of [3]; and-   (b) evaluating the pharmaceutical agent for treating or preventing    diabetic disorder for side effects;-   [12] a non-human animal model for liver cancer, which is produced by    further rearing the non-human animal of any one of [1] to [6];-   [13] the non-human animal of [12], which is structurally    characterized by the following pathological morphology:-   (a) massive type cord-like liver cell carcinoma;-   (b) infiltration of inflammatory cells; or-   (c) liver cancer caused by cirrhosis developed such that it    displaces normal liver cells;-   [14] a method of screening for a substance for treating or    preventing liver cancer, which comprises the steps of:-   (a) administering a test substance to the non-human animal model for    liver cancer of [12] or [13]; and-   (b) evaluating a therapeutic effect on liver cancer; and-   [15] a method of evaluating a medicinal substance for efficacy    against liver cancer treatment, which comprises the steps of:-   (a) administering a test medicinal substance to the non-human animal    model of liver cancer of [12] or [13]; and-   (b) evaluating a therapeutic effect on liver cancer.

Effects of the Invention

To produce experimental animals that develop pathological conditionssimilar to those of humans, insulin resistance was induced in mice, andfatty liver was induced by feeding them with a high fat diet.

Mice of different ages were sacrificed and each organ, mainly liver, wasanalyzed histopathologically (HE staining, fat staining, immunostainingfor macrophages and fibroblasts). NAFLD Activity Score (NAS; reference:“Kleiner D E et al., Hepatology. 2005 June; 41(6): 1313-21”) wascalculated to assess the pathological features in detail. Model animalsof the present invention can also be assessed for NASH by using the sameNAFLD Activity Score as for human. Thus, the model animals of thepresent invention are very useful as NASH model animals.

Furthermore, serobiochemical tests were carried out using FUJIFILMDRI-CHEM. Gene expression analysis was performed using Real-Time RT-PCR(Takara).

As described above, the present inventors successfully producedsteatohepatitis model animals (for example, NASH model animals) andliver cancer model animals that show pathological findings similar tothose of humans.

The present invention provides simple techniques for stably producinganimals that develop at an early stage a pathological condition similarto human NASH, which leads to fatty liver, steatohepatitis, hepaticfibrosis, and liver cirrhosis, followed by spontaneous development ofliver cancer as a result of progression of the pathological condition,by inducing insulin resistance in experimental animals such as mice, andloading them with a high fat diet.

Furthermore, diabetic disorders (diabetic nephritis, retinopathy,hyperlipidemia, and arteriosclerosis) can also be simultaneouslyobserved in the animals. Thus, the present invention also providestechniques for producing experimental animals that enable thepathological conditions of metabolic syndrome to be observed at the sametime.

Meanwhile, in ob/ob mice and db/db mice which are commonly used as anNASH model, the pathological condition does not develop uniformly withaging. It is necessary to monitor the pathological condition of the miceto accurately assess the disease state of NASH, which makes theexperiments cumbersome and complicated. Furthermore, the pathologicallesions are not always irreversible. This has made the efficacyassessment difficult (Horie Y et al., J din Invest 113: 1774-1783, 2004;Yanagitani A et al., Hepatology 40: 366-375, 2004; Anstee Q M et al.,Int J Exp Path 87: 1-16, 2006). Meanwhile, in the model animals of thepresent invention, the period leading to the mature pathologicalcondition is constant and its progression is irreversible. Thus, themodel animals of the present invention can be used to solve theabove-described problems.

The model animals of the present invention can be used in preclinicaltests for various therapeutic agents, and are very useful in developingagents and searching for therapeutic targets.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a result of serobiochemical test in mice of the presentinvention.

FIG. 2 shows in photographs a result of fat staining of liver from amouse of the present invention.

FIG. 3 shows in photographs a result of immunostaining of liver from amouse of the present invention.

FIG. 4 shows in photographs a result of HE staining of liver from a20-week-old mouse of the present invention.

FIG. 5 shows a result of HE staining of liver from C57BL/6J inphotographs and the NAFLD Activity Score in a graph.

FIG. 6 shows in photographs a result of F4/80 immunostaining of pancreasand adipose tissue from C57BL/6J.

FIG. 7 shows in photographs a result of immunostaining of F4/80 andER-TR7 of 8-week-old BALB/c and C3H/HeN mice.

FIG. 8 shows photographs of liver cancer in 20-week-old C3H/HeN.

FIG. 9 shows in photographs reproducible results of NASH model micereared with other high fat diets.

FIG. 10 shows in photographs and graphs results of pharmacological testsusing the NASH model mice. 1 shows the result of histological analysisin photographs. 2 shows the result of gene expression analysis inphotographs.

FIG. 11 shows the onset of diabetic complications in photographs. 1shows diabetic nephropathy in photographs, while 2 shows diabeticretinopathy (immunostaining images of new blood vessels; CD31) inphotographs.

MODE FOR CARRYING OUT THE INVENTION

The present invention relates to steatohepatitis model animals producedby inducing insulin resistance.

In a preferred embodiment, the present invention providessteatohepatitis model animals (herein sometimes referred to as “modelanimals of the present invention”) prepared by administering agents forinducing organ inflammation.

Model animals of the present invention are prepared by administering toexperimental animals agents for inducing organ inflammation and thenpreferably feeding them with high fat diets.

Animals to be used in the present invention are not particularlylimited, as long as they are commonly used as experimental animals. Suchanimals typically include non-human animals, preferably non-humanvertebrates, more preferably non-human mammals, and still morepreferably rodents. Examples of animals that can be used to preparemodel animals of the present invention specifically include mice, rats,rabbits, dogs, chickens, and monkeys (such animals are sometimes alsoreferred to simply as “experimental animals”).

The genetic background of the animals to be used to produce modelanimals of the present invention is not particularly limited; and it ispossible to use animals with any genetic background. In general,wild-type animals can be preferably used.

In the present invention, the agents for inducing organ inflammation(herein sometimes also referred to simply as “agents”) are notparticularly limited, as long as they have an activity of directly orindirectly inducing inflammation in various organs. Organs for inducinginflammation include, for example, pancreas, adipose tissues, and muscletissues. The agents for inducing organ inflammation of the presentinvention also include agents that directly or indirectly induceinflammation in the peripheral tissues.

The agents for inducing organ inflammation of the present inventionpreferably include N-acetyl-β-D-glucosaminidase inhibitors.

In a preferred embodiment, model animals of the present invention areproduced by administering N-acetyl-β-D-glucosaminidase inhibitors to theabove-described experimental animals. Such inhibitors include, forexample, streptozotocin and Pugnac.

Alternatively, nucleic acids having an activity of inhibitingN-acetyl-β-D-glucosaminidase can also be used as an agent of the presentinvention. Specifically, such nucleic acids include, for example, siRNAsthat suppress the expression of the O-G1cNAcase gene (GenBank accessionNo. NM_(—)023799.3), antisenses of the gene, and ribozymes that targetthe gene.

In producing the model animals of the present invention, the dosage formof the agents for inducing organ inflammation is not particularlylimited. For example, the dosage form includes, for example,subcutaneous administration (subcutaneous injection, etc.), intravenousadministration, oral administration, and intraperitoneal administration.

The dose of the agent to be administered is not limited, and istypically 50 to 500 μg, preferably 100 to 300 μg, and more preferably200 μg when the agent is streptozotocin.

The timing for administering the agent is as follows. The agent istypically administered one to five days after birth (neonatal period,preferably one to five days old, and more preferably two days old), andpreferably two days after birth.

As described above, insulin resistance can be induced by administeringthe agents. After administration of the agents as described above, theanimals are reared to produce model animals of the present invention. Ingeneral, the animals are preferably fed with a high fat diet. Such highfat diets include various general animal diets that are commerciallyavailable.

The major ingredients of the above-described high fat diets include, forexample, crude fat, crude protein, crude fiber, crude ash, nitrogen-freeextract, and water. The high fat diets of the present invention are notparticularly limited; however, the content of crude fat is 20% or more,and preferably 30% or more; and the ratio of fat-derived calories tototal calories is typically 50% or more, and preferably 60% or more.Ingredients to be formulated into the high fat diets include, forexample, powdered beef tallow, milk casein, powdered egg white,L-cystine, safflower oil, crystalline cellulose, maltodextrin, lactose,and sucrose. These substances are shown as an example of ingredients ofthe high fat diets, and are not necessarily contained in the diets.

The above-described high fat diets include, but are not limited to, forexample, those that have a higher content of crude fat (for example,higher by about 30% or more) than normal diets. An example of such highfat diets include those commercially available as diets for laboratoryanimals, such as High Fat Diet32 (CLEA Japan Inc.) and D12492 (ResearchDiets).

For example, when the animals are mice, the above-described high fatdiet feeding begins typically at the age of 2 to 6 weeks, preferably 3to 5 weeks, and more preferably 4 weeks. In addition, for example, inthe case of mice, the amount of high fat diet given each time is about 3to 6 g. In general, mice are preferably fed with the high fat diet forone week or more. Those skilled in the art can appropriately regulate(adjust) the amount of high fat diet depending on the type, size,weight, or such of the experimental animals to be used. Fatty liver canbe induced by feeding them with a high fat diet. Thus, in a preferredembodiment, the present invention provides steatohepatitis modelanimals, which comprise the step of inducing fatty liver byadministering to the animals agents for inducing organ inflammation andby feeding them with a high fat diet.

Animals produced by the above-described method develop symptoms ofsteatohepatitis and are useful as steatohepatitis model animals. Themodel animals of the present invention have the characteristic ofsimultaneously developing pathological conditions that are observed inanimals prepared by the methods of the present invention. It ispreferred that the model animals simultaneously develop insulinresistance and/or hepatic fibrosis. However, such pathologicalconditions are not limited to these examples.

In a preferred embodiment, the model animals of the present inventionare characteristic in that the pathological conditions of metabolicsyndrome can be observed at the same time, since diabetic disorders(diabetic nephritis, retinopathy, hyperlipidemia, and arteriosclerosis)can be observed simultaneously in model animals of the presentinvention.

Furthermore, since the model animals of the present invention have thecharacteristic that pathological condition does not recoverspontaneously, the animals can be suitably used in testing and assessingdrug efficacy.

In a preferred embodiment of the model animals of the present invention,the above-described steatohepatitis is nonalcoholic hepatitis (NASH).Specifically, the present invention provides nonalcoholic hepatitis(NASH) model animals, which are prepared by administering agents forinducing organ inflammation. The animals steadily develop in a constanttime course pathological conditions with similar progression andprognosis to those of human NASH.

In a preferred embodiment, nonalcoholic hepatitis model animals of thepresent invention have at least one (preferably, all) of the followingpathological findings:

-   (1) macrovesicular fat deposition in liver cells and liver cell    ballooning;-   (2) infiltration of inflammatory cells; and-   (3) fibrosis around mainly the central vein.

Accordingly, in a preferred embodiment, model animals of the presentinvention are structurally characterized by the above-describedpathological morphologies.

As the rearing continued, the above-described steatohepatitis modelanimals of the present invention developed liver cirrhosis which led toliver cancer. Thus, steatohepatitis model animals of the presentinvention are also useful, for example, as animals (starting materials)for producing liver cirrhosis model animals or liver cancer modelanimals Specifically, the present invention provides materials forpreparing liver cirrhosis or liver cancer model animals, which comprisesteatohepatitis model animals of the present invention.

Furthermore, methods for producing model animals of the presentinvention as described above are also included in the present invention.In a preferred embodiment, the present invention provides methods forproducing steatohepatitis model animals, which comprise the step ofadministering agents for inducing organ inflammation to non-humananimals

Furthermore, substances for treating or preventing steatohepatitis canbe screened by using model animals of the present invention.Specifically, the present invention provides methods of screening forsubstances for treating or preventing steatohepatitis, which comprisethe steps of:

-   (a) administering a test substance to a steatohepatitis model animal    of the present invention; and-   (b) evaluating its ameliorating effect on steatohepatitis.

Test substances to be used in these methods are not particularlylimited. For example, such substances include single compounds such asnatural compounds, organic compounds, inorganic compounds, proteins, andpeptides, as well as compound libraries, expression products of genelibraries, cell extracts, cell culture supernatants, products offermenting microorganisms, extracts of marine organisms, and plantextracts, but are not limited thereto.

Methods for administering test substances or medicinal substances of thepresent invention are not particularly limited; however, they can beadministered, for example, orally or by injection. When such a testsubstance is a protein, for example, a viral vector carrying a geneencoding the protein may be constructed and can be introduced into modelanimals of the present invention using their infectability.

In the step of (b), the ameliorating effect on steatohepatitis can beevaluated by determining whether steatohepatitis is ameliorated byassessing the pathological findings of the model animals.

The pathological findings of steatohepatitis include, for example, theabove-described pathological findings (pathological morphologies).Herein, “amelioration” means that the symptoms of steatohepatitis arealleviated or restored to normal. By using as an indicator thepathological findings described herein, those skilled in the art canappropriately evaluate whether the symptoms of steatohepatitis areameliorated in the model animals.

In the present invention, substances that produce the amelioratingeffect in the step of (b) above can be selected as substances fortreating or preventing steatohepatitis.

Furthermore, medicinal substances can be assessed for their efficacy inameliorating steatohepatitis by using model animals of the presentinvention. Specifically, the present invention provides methods forevaluating the efficacy of medicinal substances in amelioratingsteatohepatitis, which comprise the steps of:

-   (a) administering a test medicinal substance to a steatohepatitis    model animal of the present invention; and-   (b) evaluating its ameliorating effect on steatohepatitis.

The type of medicinal substances that can be evaluated for efficacy bythe above-described methods is not particularly limited; and suchmedicinal substances include, for example, various known pharmaceuticalagents (low-molecular-weight compounds, proteins, nucleic acids, orsuch).

When a test medicinal substance exerts an ameliorating effect onsteatohepatitis, the medicinal substance is judged to have therapeuticeffect on steatohepatitis.

Furthermore, model animals of the present invention are characterized inshowing diabetic disorders (diabetic nephritis, retinopathy, or such)which develop as a complication of diabetes, simultaneously inconjunction with steatohepatitis. Thus, model animals of the presentinvention are useful as diabetes model animals.

Specifically, the present invention provides diabetes model non-humananimals prepared by administering agents for inducing organinflammation. Agents for treating or preventing diabetic disorders(diabetic nephritis, retinopathy, or such) can be developed by usingdiabetes model animals of the present invention. For example, candidatecompounds for treating or preventing diabetic disorders can be screenedby administering test substances to diabetes model animals of thepresent invention, and evaluating their ameliorating effect on diabeticdisorders.

In a preferred embodiment, the present invention provides methods ofscreening for substance for treating or preventing diabetic disorders,which comprise the steps of:

-   (a) administering a test substance to a non-human animal model of    diabetic disorder of the present invention; and-   (b) evaluating its ameliorating effect on diabetic disorder.

Many NASH patients are also diabetes patients, and they are thought todevelop various complications. Model animals of the present inventiondevelop diabetic complications and are thus very useful as modelanimals, because risks such as side effects discovered at clinicaltrials can be evaluated at earlier stages by using the model animals.

Specifically, the present invention provides methods for evaluating theside effects risks of pharmaceutical agents by using the diabetes modelanimals of the present invention.

In a preferred embodiment, the present invention relates to methods forevaluating the risk of side effects of pharmaceutical agents fortreating or preventing diabetic disorders, which comprise the steps of:

-   (a) administering a test pharmaceutical agent to the non-human    animal model of diabetic disorder of the present invention; and-   (b) evaluating the side effects of the pharmaceutical agent for    treating or preventing diabetic disorder.

Furthermore, the present inventors for the first time discovered that asthe rearing continues, the above-described steatohepatitis model animalsof the present invention develop liver cirrhosis which leads to livercancer. Thus, the present invention provides liver cirrhosis modelanimals and liver cancer model animals which are prepared by continuingto rear the above-described steatohepatitis model animals.

In a preferred embodiment, the present invention relates to liver cancermodel animals which are prepared by continuing the rearing ofsteatohepatitis model animals prepared by administering agents forinducing organ inflammation.

The above-described steatohepatitis model animals of the presentinvention subsequently develop liver cirrhosis. Liver cancer modelanimals can be produced by further rearing of the animals. In producingsuch model animals, the period of rearing after liver cirrhosis hasdeveloped is typically, for example, 2 to 20 weeks or more, andpreferably 10 weeks or more, when the experimental animal is mouse.

Liver cancer model animals of the present invention are structurallycharacterized, for example, by at least one (preferably, all) selectedfrom the following pathological findings (pathological morphologies):

-   (a) massive type cord-like liver cell carcinoma;-   (b) infiltration of inflammatory cells; and-   (c) liver cancer caused by cirrhosis developed such that it    displaces normal liver cells.

Liver cancer model animals having the above-described characteristicsexhibit the pathological morphologies described above, and thus arestructurally different from conventional liver cancer model animalsprepared by administering chemical substances (carcinogenic substances).

Substances for treating or preventing liver cancer can be selected byusing the above-described liver cancer model animals of the presentinvention.

In a preferred embodiment, the above-described methods of the presentinvention includes methods of screening for substances for treating orpreventing liver cancer, which comprise the steps of:

-   (a) administering a test substance to a liver cancer model animal of    the present invention; and-   (b) evaluating its therapeutic effect on liver cancer.

In the above-described methods, the therapeutic effect can beappropriately evaluated, for example, using as an indicator theabove-described pathological findings of liver cancer. For example, whenmassive type cord-like liver cell carcinoma is eliminated in a modelanimal of the present invention administered with a test substance, thetest substance is judged to have therapeutic effect on liver cancer.

Furthermore, according to the present invention, medicinal substancescan be evaluated for their efficacy in liver cancer treatment by usingliver cancer model animals of the present invention. In a preferredembodiment, the methods include, for example, those comprising the stepsof:

-   (a) administering a test medicinal substance to a liver cancer model    animal of the present invention; and-   (b) evaluating its therapeutic effect on liver cancer.

All prior art documents cited herein are incorporated herein byreference.

EXAMPLES

Hereinbelow, the present invention will be described more specificallywith reference to the Examples, but it is not limited thereto.

Example 1 Preparation of NASH Model Animals and Liver Cancer ModelAnimals (a) Preparation of NASH Model Mice

Gestational C57BL6J/JJcl, C3H/HeNJcl, and BALB/cByJJcl (CLEA Japan Inc.)and C57BL6J/NCrlCrlj (Charles River Japan, Inc.) were reared and allowedto deliver. Pancreatic inflammation was induced in male mice ofC57BL6J/JcL, BALB/cByJJcl, and C3H/HeNJcl (CLEA Japan Inc.) two daysafter birth with cytotoxicity specific toN-Acetyl-beta-D-glucosaminidase (O-GlcNAcase) in pancreatic β cells (forexample, by subcutaneously administering 10 mg/ml streptozotocin (SIGMA)at 20 μl/head). Thus, insulin resistance was induced by directly orindirectly eliciting inflammation in peripheral tissues. The mice werereared with a CE-2 diet (CLEA Japan Inc.) and sterile water until fourweeks old, and ablactated when they reached the age of four weeks. Then,the mice were reared until 20 weeks old with sterile water and High FatDiet (CLEA Japan Inc.) or D12492 (Research Diets), which have a highercrude fat content (or by about 30% or more) than normal diet.

(b) Histological Assessment

After mice of different ages were fasted for 24 hours, they weresacrificed under ether anesthesia, and blood was collected. Each organwas frozen in OCT compound (Sakura Fine Technical), and then sliced intosections for pathological analysis. A serobiochemical test showed thatthe levels of fasting blood glucose, alanine aminotransferase (ALT), andneutral fat were all higher in this model as compared to the group ofnormal animals. Thus, the model animals developed insulin resistance andhyperlipemia (FIG. 1). Histologically, severe fatty liver with livercell ballooning was observed at the age of five weeks. Fat was almostcompletely eliminated from the liver at the age of eight weeks, and thusthe progression of histopathological condition was very similar to thatof human burned-out NASH (FIG. 2). At the age of six weeks, accumulationand infiltration of inflammatory cells including macrophages wasobserved in their liver, and fibrosis progressed around the central veinof liver. Result obtained by further observation of changes over timeshowed that at the age of eight weeks, central veins become connected asfibrosis progressed, and liver cirrhosis with formation of regeneratingnodules was observed at the age of ten weeks (FIG. 3). Furthermore, NASwas calculated based on the histopathological data. The result showedthat the score was 5 in average at the NASH stage. Thus, it wasdemonstrated that the pharmacological effect could be evaluated bymonitoring changes of this score (FIG. 5).

The pathogenesis of NASH in this mouse model is that pancreaticinflammation triggers chronic inflammation in peripheral tissues such asliver and adipose tissues; insulin resistance develops; and persistentsystemic inflammation leads to fatty liver (FIG. 6). Then, theregenerating nodules enlarged as the mice age. Infiltration ofinflammatory cells, increase of atypical hepatocytes, and development ofcancer to displace normal liver cells were observed at the age of 20weeks (FIG. 4). Moreover, lesions that are histologically consistentwith NASH lesion which leads to liver cancer can also be developed inmice of other lines (FIGS. 7 and 8). NASH lesions were reproducible evenif the type of high fat diet was changed (FIG. 9).

Furthermore, as a trial of NASH therapy, angiotensin receptor antagonist(ARB), which is an antihypertensive agent, was orally administered tothe model of the present invention for two weeks. Consistent with aclinical report (Georgescu E. F. et al., 15: 942), comparison of theARB-administered group and the non-treated group showed histologicalimprovement in the liver and ameliorating effects on inflammation andfibrosis as detected by genetic test, which suggested, the result ishighly similar to clinical results (FIG. 10).

Furthermore, in the mouse model of the present invention, adipose tissueinflammation is also induced at the same time and this potentiatesinsulin resistance. The resulting persistent chronic hyperglycemiacauses microangiopathy and such, leading to diabetic complications(diabetic nephropathy, retinopathy, and neuropathy). Then, pathologicallesions in other organs were studied in detail. As a result, glomerularand interstitial fibrosis which are characterized by accumulation ofinflammatory macrophages and fibroblasts within and around renalglomeruli were observed at the age of ten weeks, and thus the animalsdeveloped diabetic nephropathy (FIG. 11). Furthermore, at the age of 20weeks, neovascularization in the eyes was assessed using a CD31antibody. In the model mice of the present invention, neovascular bloodhyperplasia was observed in the retina, suggesting that the micedeveloped diabetic retinopathy (FIG. 11).

Thus, the present invention provides NASH model animals which developfatty liver leading to liver cancer. The use of such model animalsfacilitates the analysis of pathogenesis and pathological condition ofhuman NASH, and development of techniques and agents for treating humanNASH.

INDUSTRIAL APPLICABILITY

The causes of fat accumulation in the liver include alcohol, obesity,diabetes, lipid metabolism abnormality, pharmaceutical agents, andsevere malnutrition. However, the causes are roughly categorized asalcoholic and nonalcoholic. Alcoholic fatty liver leads to hepatitis,liver fibrosis, and liver cirrhosis. Meanwhile, nonalcoholic fatty liverhas been believed to be a pathological condition that does not progress.However, in the late 1990s, as the obese population increased and thedisease concept became known, it was revealed that nonalcoholic fattyliver is a high-incidence disease next to type C hepatitis and alcoholichepatitis in Europe and the United States. The pathological conditionwas reported to progress into liver cirrhosis and finally liver cancer,which drew attention to the disease. In Japan, the obese population withobesity is also steadily increasing due to genetically predisposed lowinsulin secretion, and westernized diets and lack of physical activity.Under such circumstances, the number of patients diagnosed as NASH isincreasing, and thus there is an urgent demand for developing andestablishing methods and agents for treating NASH.

The model of the present invention is highly similar to the pathologicalcondition of human NASH in terms of disease progression, and can be usedto determine the stage for analyzing the phases of insulin resistance,fatty liver, steatohepatitis, liver fibrosis, and liver cirrhosis,depending on the subject to be treated. Furthermore, by searching forpathogenic factors involved in the progression of each phase, thepresent invention can also contribute to the development of completelynew methods or agents for treating human NASH, hepatic fibrosis, andliver cirrhosis, as well as biomarkers for the diseases. In addition,the present invention is applicable to evaluate pharmacokinetics in NASHlesions. Moreover, the model of the present invention finally leads toliver cancer. Thus, the present invention enables the screening forcancer-suppressing agents or such, investigation of onset mechanism forliver cancer, and pharmaceuticals that target molecules in developmentof liver cancer.

In addition to the diseases described above, diabetic disorders can alsobe analyzed at the same time. Thus, the present invention is expected togreatly contribute to the development of methods and agents for liverdiseases, elucidation of the relationships among systemic pathologicalconditions in a subject animal, as well as development of therapeuticmethods/agents and biomarkers.

1. A non-human animal model for steatohepatitis produced byadministering an agent for inducing organ inflammation.
 2. The non-humananimal of claim 1, wherein the steatohepatitis is a non-alcoholicsteatohepatitis.
 3. A non-human animal model for diabetic disorderproduced by administering an agent for inducing organ inflammation. 4.The non-human animal of claim 1, wherein the agent for inducing organinflammation is an N-acetyl-β-D-glucosaminidase inhibitor.
 5. Thenon-human animal of claim 1, which comprises the step of inducing fattyliver by administering an agent for inducing organ inflammation to theanimal and rearing the animal with a high-fat diet.
 6. The non-humananimal of claim 1, wherein the non-human animal is a mouse.
 7. A methodof producing a non-human animal model of steatohepatitis, whichcomprises the step of inducing inflammation in an organ of the non-humananimal.
 8. A method of screening for a substance for treating orpreventing steatohepatitis, which comprises the steps of: (a)administering a test substance to the non-human animal model ofsteatohepatitis of claim 1; and (b) evaluating an ameliorating effect onsteatohepatitis.
 9. A method of evaluating a medicinal substance forefficacy against steatohepatitis amelioration, which comprises the stepsof: (a) administering a test medicinal substance to the non-human animalmodel of steatohepatitis of claim 1; and (b) evaluating an amelioratingeffect on steatohepatitis.
 10. A method of screening for a substance fortreating or preventing a diabetic disorder, which comprises the stepsof: (a) administering a test substance to the non-human animal model fora diabetic disorder of claim 3; and (b) evaluating an ameliorativeeffect on diabetic disorder.
 11. A method of evaluating the side effectsrisks of a pharmaceutical agent for treating or preventing a diabeticdisorder, which comprises the steps of: (a) administering a testpharmaceutical agent to the non-human animal model for a diabeticdisorder of claim 3; and (b) evaluating the pharmaceutical agent fortreating or preventing diabetic disorder for side effects.
 12. Anon-human animal model for liver cancer, which is produced by furtherrearing the non-human animal of claim
 1. 13. The non-human animal ofclaim 12, which is structurally characterized by the followingpathological morphology: (a) massive type cord-like liver cellcarcinoma; (b) infiltration of inflammatory cells; or (c) liver cancercaused by cirrhosis developed such that it displaces normal liver cells.14. A method of screening for a substance for treating or preventingliver cancer, which comprises the steps of: (a) administering a testsubstance to the non-human animal model for liver cancer of claim 12;and (b) evaluating a therapeutic effect on liver cancer.
 15. A method ofevaluating a medicinal substance for efficacy against liver cancertreatment, which comprises the steps of: (a) administering a testmedicinal substance to the non-human animal model of liver cancer ofclaim 12; and (b) evaluating a therapeutic effect on liver cancer. 16.The non-human animal of claim 3, wherein the agent for inducing organinflammation is an N-acetyl-β-D-glucosaminidase inhibitor.
 17. Thenon-human animal of claim 3, which comprises the step of inducing fattyliver by administering an agent for inducing organ inflammation to theanimal and rearing the animal with a high-fat diet.
 18. The non-humananimal of claim 3, wherein the non-human animal is a mouse.
 19. Anon-human animal model for liver cancer, which is produced by furtherrearing the non-human animal of claim
 3. 20. The non-human animal ofclaim 19, which is structurally characterized by the followingpathological morphology; (a) massive type cord-like liver cellcarcinoma; (b) infiltration of inflammatory cells; or (c) liver cancercaused by cirrhosis developed such that it displaces normal liver cells.21. A method of screening for a substance for treating or preventingliver cancer, which comprises the steps of: (a) administering a testsubstance to the non-human animal model for liver cancer of claim 19;and (b) evaluating a therapeutic effect on liver cancer.
 22. A method ofevaluating a medicinal substance for efficacy against liver cancertreatment, which comprises the steps of: (a) administering a testmedicinal substance to the non-human model of liver cancer of claim 19;and (b) evaluating a therapeutic effect on liver cancer.